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A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA)

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Abstract

In the genome of Bacillus megaterium DSM319, a strain who has recently been sequenced to fully exploit its potential for biotechnological purposes, we identified a gene encoding the cytochrome P450 CYP106A1 as well as genes encoding potential redox partners of CYP106A1. We cloned, expressed, and purified CYP106A1 and five potential autologous redox partners, one flavodoxin and four ferredoxins. The flavodoxin and three ferredoxins were able to support the activity of CYP106A1 displaying the first cloned natural redox partners of a cytochrome P450 from B. megaterium. The CYP106A1 system was able to convert the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) belonging to the main bioactive constituents of Boswellia serrata gum resin extracts, which are used to treat inflammatory disorders and arthritic diseases. In order to provide sufficient amounts of the KBA products to characterize them structurally by NMR spectroscopy, recombinant whole-cell biocatalysts were constructed based on B. megaterium MS941. The main product has been identified as 7β-hydroxy-KBA, while the side product (∼20 %) was shown to be a mixture of 7β,15α-dihydroxy-KBA and 15α-hydroxy-KBA. Without further optimization 560.7 mg l−1 day−1 of the main product, 7β-hydroxy-KBA, could be obtained thus providing a suitable starting point for the efficient production of modified KBA by chemical tailoring to produce novel KBA derivatives with increased bioavailability and this way more efficient drugs.

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References

  • Barg H, Malten M, Jahn M, Jahn D (2005) Protein and vitamin production in Bacillus megaterium, vol 18. Microbial processes and products, 1st edn. Humana, Totowa, pp 205–223

  • Barnes HJ, Arlotto MP, Waterman MR (1991) Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli. Proc Natl Acad Sci USA 88:5597–5601

    Article  CAS  PubMed  Google Scholar 

  • Bell SG, Hoskins N, Xu F, Caprotti D, Rao Z, Wong LL (2006) Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris. Biochem Biophys Res Commun 342:191–196

    Article  CAS  PubMed  Google Scholar 

  • Berg A, Gustafsson JA, Ingelman-Sundberg M (1976) Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium. J Biol Chem 251:2831–2838

    CAS  PubMed  Google Scholar 

  • Berg A, Ingelman-Sundberg M, Gustafsson JA (1979) Isolation and characterization of cytochrome P-450meg. Acta Biol Med Ger 38:333–344

    CAS  PubMed  Google Scholar 

  • Bernhardt R (1996) Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev Physiol Biochem Pharmacol 127:137–221

    Article  CAS  PubMed  Google Scholar 

  • Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145

    Article  CAS  PubMed  Google Scholar 

  • Bernhardt R, Waterman M (2007) Cytochrome P450 and steroid hormone biosynthesis. In: Sigel A, Sigel H, Sigel RKO (eds) The ubiquitous roles of cytochrome P450 proteins. Wiley, New York, pp 361–396

    Chapter  Google Scholar 

  • Bleif S, Hannemann F, Zapp J, Hartmann D, Jauch J, Bernhardt R (2012) A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-beta-boswellic acid (KBA) based on a recombinant cytochrome P450 system. Appl Microbiol Biotechnol 93:1135–1146

    Article  CAS  PubMed  Google Scholar 

  • Chun Y, Shimada T, Sanchez-Ponce R, Martin MV, Lei L, Zhao B, Kelly SL, Waterman MR, Lamb DC, Guengerich FP (2007) Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2). J Biol Chem 282:17486–17500

    Article  CAS  PubMed  Google Scholar 

  • Eppinger M, Bunk B, Johns MA, Edirisinghe JN, Kutumbaka KK, Koenig SSK, Creasy HH, Rosovitz MJ, Riley DR, Daugherty S, Martin M, Elbourne LDH, Paulsen I, Biedendieck R, Braun C, Grayburn S, Dhingra S, Lukyanchuk V, Ball B, Ul-Qamar R, Seibel J, Bremer E, Jahn D, Ravel J, Vary PS (2011) Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. J Bacteriol 193:4199–4213

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ewen KM, Schiffler B, Takasaki Y, Bernhardt R, Hannemann F (2008) The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe. FEMS Yeast Res 8:432–441

    Article  CAS  PubMed  Google Scholar 

  • Ewen KM, Hannemann F, Khatri Y, Perlova O, Kappl R, Krug D, Hüttermann J, Müller R, Bernhardt R (2009) Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450. J Biol Chem 284:28590–28598

    Article  CAS  PubMed  Google Scholar 

  • Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acids Res 38:D211–D222

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Freigang J, Diederichs K, Schäfer KP, Welte W, Paul R (2002) Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori. Protein Sci 11:253–261

    Article  CAS  PubMed  Google Scholar 

  • Girhard M, Klaus T, Khatri Y, Bernhardt R, Urlacher VB (2010) Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis. Appl Microbiol Biotechnol 82:595–607

    Article  Google Scholar 

  • Goni G, Zöllner A, Lisurek M, Velazquez-Campoy A, Pinto S, Gomez-Moreno C, Hannemann F, Bernhardt R, Medina M (2009) Cyanobacterial electron carrier proteins as electron donors to CYP106A2 from Bacillus megaterium ATCC 13368. Biochim Biophys Acta 1794:1635–1642

    Article  CAS  PubMed  Google Scholar 

  • Green AJ, Munro AW, Cheesman MR, Reid GA, von Wachenfeldt C, Chapman SK (2003) Expression, purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI. J Inorg Biochem 93:92–99

    Article  CAS  PubMed  Google Scholar 

  • Grinberg AV, Hannemann F, Schiffler B, Müller J, Heinemann U, Bernhardt R (2000) Adrenodoxin: structure, stability, and electron transfer properties. Proteins 40:590–612

    Article  CAS  PubMed  Google Scholar 

  • Gupta I, Parihar A, Malhotra P, Gupta S, Ludtke R, Safayhi H, Ammon HP (2001) Effects of gum resin of Boswellia serrata in patients with chronic colitis. Planta Med 67:391–395

    Article  CAS  PubMed  Google Scholar 

  • Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta 1770:330–344

    Article  CAS  PubMed  Google Scholar 

  • Hawkes DB, Adams GW, Burlingame AL, Ortiz de Montellano PR, De Voss JJ (2002) Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization. J Biol Chem 277:27725–27732

    Article  CAS  PubMed  Google Scholar 

  • He JS, Fulco AJ (1991) A barbiturate-regulated protein binding to a common sequence in the cytochrome P450 genes of rodents and bacteria. J Biol Chem 266:7864–7869

    CAS  PubMed  Google Scholar 

  • Jauch J, Bergmann J (2003) An efficient method for the large-scale preparation of 3-O-acetyl-11-oxo-β-boswellic acid and other boswellic acids. Eur J Org Chem 2003:4752–4756

    Article  Google Scholar 

  • Jenkins CM, Waterman MR (1994) Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. J Biol Chem 269:27401–27408

    CAS  PubMed  Google Scholar 

  • Khatri Y, Hannemann F, Ewen KM, Pistorius D, Perlova O, Kagawa N, Brachmann AO, Müller R, Bernhardt R (2010) The CYPome of Sorangium cellulosum So ce56 and identification of CYP109D1 as a new fatty acid hydroxylase. Chem Biol 17:1295–1305

    Article  CAS  PubMed  Google Scholar 

  • Knight E Jr, D'Eustachio AJ, Hardy RW (1966) Flavodoxin: a flavoprotein with ferredoxin activity from Clostrium pasteurianum. Biochim Biophys Acta 113:626–628

    Article  CAS  PubMed  Google Scholar 

  • Krüger P, Daneshfar R, Eckert GP, Klein J, Volmer DA, Bahr U, Müller WE, Karas M, Schubert-Zsilavecz M, Abdel-Tawab M (2008) Metabolism of boswellic acids in vitro and in vivo. Drug Metab Dispos 36:1135–1142

    Article  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lampe JN, Fernandez C, Nath A, Atkins WM (2008) Nile Red is a fluorescent allosteric substrate of cytochrome P450 3A4. Biochemistry 47:509–516

    Article  CAS  PubMed  Google Scholar 

  • Lawson RJ, von Wachenfeldt C, Haq I, Perkins J, Munro AW (2004) Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: biophysical properties and interactions with cytochrome P450 BioI. Biochemistry 43:12390–12409

    Article  CAS  PubMed  Google Scholar 

  • Malca SH, Girhard M, Schuster S, Dürre P, Urlacher VB (2011) Expression, purification and characterization of two Clostridium acetobutylicum flavodoxins: potential electron transfer partners for CYP152A2. Biochim Biophys Acta 1814:257–264

    Article  PubMed  Google Scholar 

  • Malten M, Hollmann R, Deckwer WD, Jahn D (2005) Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium. Biotechnol Bioeng 89:206–218

  • McLean KJ, Warman AJ, Seward HE, Marshall KR, Girvan HM, Cheesman MR, Waterman MR, Munro AW (2006) Biophysical characterization of the sterol demethylase P450 from Mycobacterium tuberculosis, its cognate ferredoxin, and their interactions. Biochemistry 45:8427–8443

    Article  CAS  PubMed  Google Scholar 

  • Meinhardt F, Stahl U, Ebeling W (1989) Highly efficient expression of homologous and heterologous genes in Bacillus megaterium. Appl Microbiol Biotechnol 30:343–350

    Article  CAS  Google Scholar 

  • Nguyen KT, Virus C, Günnewich N, Hannemann F, Bernhardt R (2012) Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone. ChemBioChem 13:1161–1166

    Article  CAS  PubMed  Google Scholar 

  • Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378

    CAS  PubMed  Google Scholar 

  • Poeckel D, Werz O (2006) Boswellic acids: biological actions and molecular targets. Curr Med Chem 13:3359–3369

    Article  CAS  PubMed  Google Scholar 

  • Puan K, Wang H, Dairi T, Kuzuyama T, Morita CT (2005) fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. FEBS Lett 579:3802–3806

    Article  CAS  PubMed  Google Scholar 

  • Rabe KS, Gandubert VJ, Spengler M, Erkelenz M, Niemeyer CM (2008) Engineering and assaying of cytochrome P450 biocatalysts. Anal Bioanal Chem 392:1059–1073

    Article  CAS  PubMed  Google Scholar 

  • Richhardt J, Larsen M, Meinhardt F (2010) An improved transconjugation protocol for Bacillus megaterium facilitating a direct genetic knockout. Appl Microbiol Biotechnol 86:1959–1965

    Article  CAS  PubMed  Google Scholar 

  • Safayhi H, Rall B, Sailer ER, Ammon HP (1997) Inhibition by boswellic acids of human leukocyte elastase. J Pharmacol Exp Ther 281:460–463

    CAS  PubMed  Google Scholar 

  • Sancho J (2006) Flavodoxins: sequence, folding, binding, function and beyond. Cell Mol Life Sci 63:855–864

    Article  CAS  PubMed  Google Scholar 

  • Schmitz D, Zapp J, Bernhardt R (2012) Hydroxylation of the triterpenoid dipterocarpol with CYP106A2 from Bacillus megaterium. FEBS J 279:1663–1674

    Article  CAS  PubMed  Google Scholar 

  • Sheng H, Sun H (2011) Synthesis, biology and clinical significance of pentacyclic triterpenes: a multi-target approach to prevention and treatment of metabolic and vascular diseases. Nat Prod Rep 28:543

    Article  CAS  PubMed  Google Scholar 

  • Sielaff B, Andreesen JR (2005) Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5. FEBS J 272:1148–1159

    Article  CAS  PubMed  Google Scholar 

  • Siemoneit U, Hofmann B, Kather N, Lamkemeyer T, Madlung J, Franke L, Schneider G, Jauch J, Poeckel D, Werz O (2008) Identification and functional analysis of cyclooxygenase-1 as a molecular target of boswellic acids. Biochem Pharmacol 75:503–513

    Article  CAS  PubMed  Google Scholar 

  • Smillie RM (1965) Isolation of two proteins with chloroplast ferredoxin activity from a blue-green alga. Biochem Biophys Res Commun 20:621–629

    Article  CAS  PubMed  Google Scholar 

  • Sticht H, Rösch P (1998) The structure of iron-sulfur proteins. Prog Biophys Mol Biol 70:95–136

    Article  CAS  PubMed  Google Scholar 

  • Sussman MD, Vary PS, Hartman C, Setlow P (1988) Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease. J Bacteriol 170:4942–4945

    CAS  PubMed Central  PubMed  Google Scholar 

  • Syrovets T, Büchele B, Gedig E, Slupsky JR, Simmet T (2000) Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIalpha. Mol Pharmacol 58:71–81

    CAS  PubMed  Google Scholar 

  • Syrovets T, Büchele B, Krauss C, Laumonnier Y, Simmet T (2005) Acetyl-boswellic acids inhibit lipopolysaccharide-mediated TNF-alpha induction in monocytes by direct interaction with IkappaB kinases. J Immunol 174:498–506

    CAS  PubMed  Google Scholar 

  • Tausch L, Henkel A, Siemoneit U, Poeckel D, Kather N, Franke L, Hofmann B, Schneider G, Angioni C, Geisslinger G, Skarke C, Holtmeier W, Beckhaus T, Karas M, Jauch J, Werz O (2009) Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense. J Immunol 183:3433–3442

    Article  CAS  PubMed  Google Scholar 

  • Urlacher VB, Eiben S (2006) Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol 24:324–330

    Article  CAS  PubMed  Google Scholar 

  • Urlacher VB, Girhard M (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol 30:26–36

    Article  CAS  PubMed  Google Scholar 

  • Vary PS, Biedendieck R, Fuerch T, Meinhardt F, Rohde M, Deckwer WD, Jahn D (2007) Bacillus megaterium-from simple soil bacterium to industrial protein production host. Appl Microbiol Biotechnol 76:957–967

    Article  CAS  PubMed  Google Scholar 

  • Virus C, Lisurek M, Simgen B, Hannemann F, Bernhardt R (2006) Function and engineering of the 15beta-hydroxylase CYP106A2. Biochem Soc Trans 34:1215–1218

    Article  CAS  PubMed  Google Scholar 

  • Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol. 1:REVIEWS3003

  • Werz O, Siemoneit U, Henkel A, Jauch J, Kather N Use of boswellic acids and synthetic boswellic acid derivatives for inhibiting microsomal prostaglandin E2 synthase and cathepsin G. Ger. Offen. (2009) DE 102008015607 A1 20091015

  • Whitehouse CJC, Bell SG, Wong LL (2012) P450(BM3) (CYP102A1): connecting the dots. Chem Soc Rev 41:1218–1260

    Article  CAS  PubMed  Google Scholar 

  • Wittchen KD, Meinhardt F (1995) Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl Microbiol Biotechnol 42:871–877

    Article  CAS  PubMed  Google Scholar 

  • Zehentgruber D, Hannemann F, Bleif S, Bernhardt R, Lütz S (2010) Towards preparative scale steroid hydroxylation with cytochrome P450 monooxygenase CYP106A2. ChemBioChem 11:713–721

    Article  CAS  PubMed  Google Scholar 

  • Zheng M, Doan B, Schneider TD, Storz G (1999) OxyR and SoxRS regulation of fur. J Bacteriol 181:4639–4643

    CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

The authors thank Prof. Dr. Dieter Jahn (TU Braunschweig) for the kind provision of the B. megaterium strain MS941 and Dr. Rebekka Biedendieck (TU Braunschweig) for constructive discussions.

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Authors and Affiliations

  1. Department of Biochemistry, Saarland University, 66123, Saarbruecken, Germany

    Elisa Brill, Frank Hannemann & Rita Bernhardt

  2. Department of Pharmaceutical Biology, Saarland University, 66123, Saarbruecken, Germany

    Josef Zapp

  3. Department of Organic Chemistry II, Saarland University, 66123, Saarbruecken, Germany

    Gerit Brüning & Johann Jauch

  4. Institut für Biochemie, Universität des Saarlandes, Campus B.2.2, 66123, Saarbruecken, Germany

    Rita Bernhardt

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  1. Elisa Brill
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  2. Frank Hannemann
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Correspondence to Rita Bernhardt.

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Brill, E., Hannemann, F., Zapp, J. et al. A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA). Appl Microbiol Biotechnol 98, 1703–1717 (2014). https://doi.org/10.1007/s00253-013-5029-0

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